Methods Plasmid constructs The vesicular stomatitis virus glycoprotein expres sion vector pHITG, the HIV 1 proviral construct pNL4 3, pNL ADA, and the HIV 1 proviral indi cator constructs pNL Luc E and Bosutinib IC50 pNL Luc E R have been described previously. To introduce D64A muta tion into IN to cre ate pNL IN D64A, site directed mutagenesis was performed using pNL4 3 as a template. To create pNL ADA IN D64A and pNL Luc IN D64A E that contained D64A mutants, the SpeI PflMI fragment of pNL IN D64A was replaced with those of pNL ADA and pNL Luc E, respectively. To create the Vpr deficient construct pNL ADA R, pNL ADA IN D64A R, and pNL Luc IN D64A E R, the PflMI SalI fragment of pNL Luc E R was replaced with those of pNL ADA, pNL ADA IN D64A, and pNL Luc IN D64A E, respectively.

The neomycin resistant marker expressing vector pNL Neo E R was created by inserting a PCR amplified neomycin resistant gene into the NotI XhoI site of pNL Luc E R. To create a neomycin resistant marker expressing D64A, the Inhibitors,Modulators,Libraries mutant pNL Neo IN D64A E R was created by the SpeI PflMI fragment of pNL IN D64A and replaced with that of pNL Neo E R. To create pIRES2 EGFP I SceI, a pIRES2 EGFP based plasmid with an Inhibitors,Modulators,Libraries I SceI recognition site, a synthetic double stranded oligonucleo tide was inserted into the EcoRI and BamHI sites of pIRES2 EGFP. To make the adenoviral vector Ad I PpoI, I PpoI cDNA was amplified from pBabe HA ER I PpoI using the Adeno PpoI DraI F and Adeno PpoI DraI R primers and cloned into the SwaI site of the pAxCALNLwtit2 cosmid vector.

To generate the EGFP expressing lentiviral vector, EGFP cDNA from pENTR1a EGFP was cloned into pLenti6V5 DEST using LR Clonase. The IN D64V mutation of the gag pol expressing plasmid pLP1 was introduced using pLP1 as a template with site directed Inhibitors,Modulators,Libraries mutagenesis. Inhibitors,Modulators,Libraries Cell culture cell lines were obtained from the RIKEN Cell Bank. TIG 3 and MT 4 cells were obtained from the Health Science Research Resources Bank. HT1080, HEK293, HEK293T, MAGIC5, and TIG 3 cells were maintained in Dulbeccos modified Inhibitors,Modulators,Libraries Eagles medium supplemented with 10% fetal bovine serum. MT 4 cell was maintained in RPMI 1640 supplemented with 10% FBS. To obtain macrophage like cells, THP 1 cells, maintained in Iscoves modified Dulbeccos medium supplemented with 10% FBS, were treated for 2 d with 5. 0 10 8 M PMA. As described previ ously, PMA treated THP 1 cells were positive for Mac 1, a specific marker of macrophages.

Peripheral blood was derived from healthy donors who worked within the institute and gave informed consent. Experimental procedures were approved by the internal review board. PBMCs and MDMs were prepared and cultured as previ customer review ously described. MDMs were prepared from healthy volunteers who gave informed consents. The experimental protocol was approved by the internal review board. HIV 1 and lentiviral vector preparation The preparation and titration of replication competent and VSVG pseudotyped viruses are described elsewhere.